Determination of the binding behavior of cell membrane receptor proteins toward natural or artificial ligands is important for many biological and medical studies. In the field of target or drug discovery, high throughput screening efforts are key to isolating target-specific binders, agonists, or antagonists. Many of these therapeutic or diagnostic targets are cell surface antigens that, upon recognition by natural or synthetic binding molecules, trigger a network of signal transduction and gene regulation events inside the cell that result in cellular responses important in the initiation or maintenance of a disease. Target antigens may also differentially reside on the surface of cells and signify a unique state of physiology or disease progress in the tissue or organ. In researching these cell surface targets, the isolation of target-specific binders provides an invaluable tool for detection and perturbation at the molecular level.
Assays for cell surface receptors have normally been based on a ligand-receptor interaction in which the ligand is labeled and the amount of labeled ligand bound to the receptor is measured. Types of assays include screening for monolconal antibodies against biomarkers and therapeutic targets specific for diseased cells that are not found on normal cells, and screening for ligands or pharmacological agents specific for surface antigens that are endogenously expressed or over-expressed as recombinant proteins on different cell lines. Such assays often involve complicated procedures of separating the receptor proteins to be assayed from cellular material and therefore cannot be done in realtime. Further, it may be necessary to label several kinds of ligands or receptors, because receptors are normally specific to only one antigenic determinant.
WO 01/42489 describes a method for fluorescence detection of lipid membranes using lipophilic functionalized nanocrystals to label the membranes. The labeled membranes are then exposed to an excitation light source, and fluorescence emitted from the excited nanocrystals is detected. In a photodynamic therapy method of U.S. Pat. No. 6,375,930, a photosensitive texaphyrin is conjugated to estradiol or cholesterol. Red blood cells or white blood cells, are then loaded with the conjugates. The complex, upon exposure to light, ruptures depositing its contents.
However, there are several drawbacks to using fluorescent substrates for the analysis of cells. For example, it is difficult to get the substrate through the outer cell membrane without disrupting the cell. Moreover, there is the problem of cell leakage. Thus, following enzymatic hydrolysis of fluorescein di-62-galactopyranoside, the resulting fluorescein rapidly leaks out of the cell, with a half life of about 10 minutes. There remains a need, therefore, for compositions and methods for labeling membranes.